The next generation of medical devices and engineered tissues will require development of scaffolds that mimic the structural and functional properties of the extracellular matrix (ECM) component of tissues. Unfortunately, little is known regarding how ECM microstructure participates in the transmission of mechanical load information from a global (tissue or construct) level to a level local to the resident cells ultimately initiating relevant mechanotransduction pathways. In this study, the transmission of mechanical strains at various functional levels was determined for three-dimensional (3D) collagen ECMs that differed in fibril microstructure. Microstructural properties of collagen ECMs (e.g., fibril density, fibril length, and fibril diameter) were systematically varied by altering in vitro polymerization conditions. Multiscale images of the 3D ECM macro- and microstructure were acquired during uniaxial tensile loading. These images provided the basis for quantification and correlation of strains at global and local levels. Results showed that collagen fibril microstructure was a critical determinant of the 3D global and local strain behaviors. Specifically, an increase in collagen fibril density reduced transverse strains in both width and thickness directions at both global and local levels. Similarly, collagen ECMs characterized by increased fibril length and decreased fibril diameter exhibited increased strain in width and thickness directions in response to loading. While extensional strains measured globally were equivalent to applied strains, extensional strains measured locally consistently underpredicted applied strain levels. These studies demonstrate that regulation of collagen fibril microstructure provides a means to control the 3D strain response and strain transfer properties of collagen-based ECMs.